Auto-Antibody Production During Experimental Atherosclerosis in Mice.

Current models stipulate that B cells and antibodies function during atherosclerosis in two distinct ways based on antibody isotype, where IgM is protective and IgG is inflammatory. To examine this model, we generated mice, which are unable to produce IgG antibodies due to the absence of activation-induced deaminase (AID) but maintain high plasma cholesterol due to the absence of apolipoprotein E (APOE). We saw a dramatic decrease in plaque formation in mice compared to mice.

DNA Breaks in Ig V Regions Are Predominantly Single Stranded and Are Generated by UNG and MSH6 DNA Repair Pathways.

Antibody diversity is initiated by activation-induced deaminase (AID), which deaminates cytosine to uracil in DNA. Uracils in the Ig gene loci can be recognized by uracil DNA glycosylase (UNG) or mutS homologs 2 and 6 (MSH2-MSH6) proteins, and then processed into DNA breaks. Breaks in switch regions of the H chain locus cause isotype switching and have been extensively characterized as staggered and blunt double-strand breaks.

Spectral Properties of Fluorogenic Thiophene-derived Triarylmethane Dyes.

Spectral properties and fluorogenic behaviors of five novel thiophene variants of malachite green (MG), termed MGTs, were determined. Appreciable changes as a function of homologation and substitution pattern, including absorption band positions and intensities and fluorescence quantum yields were observed. In particular, the shorter wavelength y-band absorption was found to shift over a nearly 200 nm range based on aryl group variation, allowing fine-tuning of the excitation wavelength for these dyes.

Antibody diversification caused by disrupted mismatch repair and promiscuous DNA polymerases.

The enzyme activation-induced deaminase (AID) targets the immunoglobulin loci in activated B cells and creates DNA mutations in the antigen-binding variable region and DNA breaks in the switch region through processes known, respectively, as somatic hypermutation and class switch recombination. AID deaminates cytosine to uracil in DNA to create a U:G mismatch. During somatic hypermutation, the MutSα complex binds to the mismatch, and the error-prone DNA polymerase η generates mutations at A and T bases.

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules.

Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5.

ATAD5 deficiency decreases B cell division and Igh recombination.

Mammalian ATPase family AAA domain-containing protein 5 (ATAD5) and its yeast homolog enhanced level of genomic instability 1 are responsible for unloading proliferating cell nuclear antigen from newly synthesized DNA. Prior work in HeLa and yeast cells showed that a decrease in ATAD5 protein levels resulted in accumulation of chromatin-bound proliferating cell nuclear antigen, slowed cell division, and increased genomic instability. In this study, B cells from heterozygous (Atad5(+/m)) mice were used to examine the effects of decreased cell proliferation on Ab diversity.

Synthesis and characterization of conformationally preorganized, (R)-diethylene glycol-containing γ-peptide nucleic acids with superior hybridization properties and water solubility.

Developed in the early 1990s, peptide nucleic acid (PNA) has emerged as a promising class of nucleic acid mimic because of its strong binding affinity and sequence selectivity toward DNA and RNA and resistance to enzymatic degradation by proteases and nucleases; however, the main drawbacks, as compared to other classes of oligonucleotides, are water solubility and biocompatibility. Herein we show that installation of a relatively small, hydrophilic (R)-diethylene glycol ("miniPEG", R-MP) unit at the γ-backbone transforms a randomly folded PNA into a right-handed helix. Synthesis of optically pure (R-MP)γPNA monomers is described, which can be accomplished in a few simple steps from a commercially available and relatively cheap Boc-l-serine.

Blue fluorescent dye-protein complexes based on fluorogenic cyanine dyes and single chain antibody fragments.

Fluoromodules are complexes formed upon the noncovalent binding of a fluorogenic dye to its cognate biomolecular partner, which significantly enhances the fluorescence quantum yield of the dye. Previously, several single-chain, variable fragment (scFv) antibodies were selected from a yeast cell surface-displayed library that activated fluorescence from a family of unsymmetrical cyanine dyes covering much of the visible and near-IR spectrum. The current work expands our repertoire of genetically encodable scFv-dye pairs by selecting and characterizing a group of scFvs that activate fluorogenic violet-absorbing, blue-fluorescing cyanine dyes, based on oxazole and thiazole heterocycles.

Enhanced photostability of genetically encodable fluoromodules based on fluorogenic cyanine dyes and a promiscuous protein partner.

Fluoromodules are discrete complexes of biomolecules and fluorogenic dyes. Binding of the dyes to their cognate biomolecule partners results in enhanced dye fluorescence. We exploited a previously reported promiscuous binding interaction between a single-chain, variable fragment antibody protein and a family of cyanine dyes to create new protein-dye fluoromodules that exhibit enhanced photostability while retaining high affinity protein-dye binding.

Thermodynamics of the fragile X mental retardation protein RGG box interactions with G quartet forming RNA.

Fragile X syndrome, the most common form of inherited mental retardation, is the result of an unstable expansion of a CGG trinucleotide repeat in the 5' UTR of the fragile X mental retardation-1 (FMR1) gene. The abnormal hypermethylation of the expanded CGG repeats causes the transcriptional silencing of the FMR1 gene and, consequently, the loss of the fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein that binds to G quartet forming RNA using its RGG box motif.